The present invention relates to a poly(A)-specific 3xe2x80x2-exonuclease activity which can be obtained by chromatographically purifying a crude extract of animal or human cells and to its use for deadenylating 3xe2x80x2-poly(A) tails of nucleic acids and as a pharmaceutical or diagnostic agent, or for identifying functional interactors.
Most eukaryotic mRNAs carry poly(A) tails of approx. 200 adenosine residues in length at their 3xe2x80x2 ends. These poly(A) tails appear to influence not only the half-life or intracellular transport of mRNAs but also translation of the mRNA into the corresponding protein. While the precise mechanism has still not been elucidated, the synthesis and degradation of poly(A) tails appear to be directly or indirectly connected with the function of the tails (see, e.g., Wickens, M. et al. (1977) Curr. Opin. Genet. Dev., 7, 220-232).
Poly(A)-degrading nuclease activities have already been investigated in several eukaryotic systems (see, e.g., Virtanen, A. and xc3x85strxc3x6m, J. (1997) in Prog. Mol. Subcell. Biol. (Jeanteur, P., ed.) Vol. 16, 199-220, Springer-Verlag, Berlin-Heidelberg). Thus, two reaction pathways have, for example, been identified in yeasts. One of the reaction pathways, i.e. the so-called deadenylation dependent decapping passway, is started by removal of the poly(A) tail and concerns a 5xe2x80x2-3xe2x80x2-exonucleolytic degradation of mRNA by an Xm1p5xe2x80x2-exonuclease. The other reaction pathway, i.e. the so-called 3xe2x80x2-5xe2x80x2-decay passway, is started by a deadenylation of the mRNA and concerns a 3xe2x80x2-5xe2x80x2-exoribonucleolytic degradation of mRNA. A multicomponent complex, termed an exosome, has recently been identified as being involved in the 3xe2x80x2-5xe2x80x2-decay passway (Mitchell, P. et al. (1997), Cell 91, 457-466). The exosome consists of several 3xe2x80x2-5xe2x80x2-exoribonucleases and is involved both in 5.8S rRNA 3xe2x80x2 processing and in the 3xe2x80x2-5xe2x80x2 degradation of mRNA (Anderson, J. S. J. and Parker, R. (1998) EMBO J. 17, 1497-1506). However, it is not known whether any of the exoribonucleases of the exosome degrade poly(A) preferentially. In addition to this, a poly(A)-binding protein I (PABI)-dependent poly(A)-specific nuclease (PAN) has been identified in yeasts (see, e.g., Lowell, J. E. et al. (1992) Genes Dev. 6, 2088-2099). PAN is a 3xe2x80x2-5xe2x80x2-exoribonuclease and is composed of at least two polypeptides, i.e. Pan2p and Pan3p (see, e.g., Brown, C. E. Jr. et al. (1996) Mol. Cell. Biol., 16, 5744-5753).
At least three different poly(A)-degrading activities have been characterized in mammalian cells. For example, an activity found in Hela cells has a high selectivity for degrading 3xe2x80x2-located poly(A) tails, requires 3xe2x80x2-located hydroxyl groups and forms 5xe2x80x2-AMP as the mononucleotide reaction product (see, e.g., xc3x85strxc3x6m, J. et al. (1992) J. Biol. Chem. 267 (25), 18154-18159 and J. Astrxc3x6m, A. Astrxc3x6m and A. Virtanen, In vitro deadenylation of mammalian mRNA by a HeLa cell 3xe2x80x2 exonuclease, EMBO J., (1991), Vol 10, 3067). Furthermore, an Mg2+-dependent poly(A)-specific 3xe2x80x2-exoribonuclease having a molecular weight of 74 kDa (Kxc3x6rner, C. G. and Wahle, E. (1997) J. Biol. Chem., 272 (16), 10448-10456) has been described in calf thymus. A polyribosome-associated 3xe2x80x2-exoribonuclease having a molecular weight of 33 kDa has also been described, but this 3xe2x80x2-exoribonuclease is not specific for poly(A) (Caruccio, N. and Ross, J. (1994) J. Biol. Chem. 269 (50), 31814-31821). Another poly(A)-specific 3xe2x80x2-exonuclease activity having a molecular weight of 60 kDa was identified in calf thymus. However, it subsequently turned out that this protein is the hnRNP L protein and consequently has no connection with the poly(A)-specific 3xe2x80x2-exoribonuclease activity which has been measured.
The object of the present invention was therefore to make available a 3xe2x80x2-exoribonuclease which specifically degrades 3xe2x80x2-located poly(A) tails.
The present invention therefore relates to a process for isolating a poly(A)-specific 3xe2x80x2-exonuclease activity, which process contains the following steps:
a) preparing a crude extract from animal or human cells;
b) precipitating the protein present in the crude extract obtainable from step (a);
c) subjecting the precipitate obtainable from step (b) to chromatography on a basic anion exchanger;
d) subjecting the active fractions from step (c) to affinity chromatography;
e) subjecting the active fractions from step (d) to chromatography on a basic anion exchanger;
f) subjecting the active fractions from step (e) to affinity chromatography;
g) subjecting the active fractions from step (f) to poly(A)-affinity chromatography;
h) subjecting the active fractions from step (g) to chromatography on a basic anion exchanger; and, where appropriate,
i) subjecting the active fractions from step (g) to gel filtration. Or
j) subjecting the active fractions from step (f) to two rounds of affinity chromatography;
Surprisingly, the 3xe2x80x2-exonuclease activity which can be obtained by the above-described process is specific for 3xe2x80x2-located poly(A) tails, with a 3xe2x80x2-located hydroxyl group being required and with 5xe2x80x2-AMP being formed as the mononucleotide reaction product. In contrast to the already known 3xe2x80x2-exonuclease activities, the 3xe2x80x2-exonuclease activity according to the invention has a molecular weight of approx. 50 kDa under denaturing conditions. In contrast to the 74 kDa protein from calf thymus, the 3xe2x80x2-exonuclease activity according to the invention is not stimulated by spermidine at low salt concentrations; on the contrary, if anything, it is inhibited both at low and at high salt concentrations. Furthermore, in contrast to the calf thymus 74 kDa protein, the 3xe2x80x2-exonuclease according to the invention interacts relatively strongly with Heparin Sepharose. Furthermore, the calf thymus 74 kDa protein is not found in the SDS-Pagexe2x80x94FIGS. 1B and 2B. In addition, it was surprising that, in contrast to the HeLa cell exonuclease activity, the exonuclease according to the invention is also active in the presence of Mn2+. It is also surprising that the exonuclease according to the invention operates progressively, i.e. the exonuclease binds to the 3xe2x80x2 end of the poly(A) tail and degrades it nucleotide by nucleotide without the exonuclease-poly(A) complex dissociating, whereas the 74 kDa protein operates distributively, with the complex dissociating after one operational step and having to be regenerated.
The poly(A)-specific 3xe2x80x2-exonuclease activity according to the invention can preferably be isolated from animal or human thymus cells, in particular from calf thymus. In general, a whole cell extract is prepared for this purpose, with protein preferably being precipitated from this extract with ammonium sulfate. In this connection, preference is given to the saturation concentration being approx. 45% ammonium sulfate. It has been found to be particularly advantageous in this connection if, before the true protein precipitation, foreign proteins are separated off by being precipitated at a saturation concentration of preferably approx. 25% ammonium sulfate, such that the desired 3xe2x80x2-exonuclease activity can, in a subsequent step, be precipitated out of the supernatant at an ammonium sulfate saturation of approx. 45%. This protein fractionation itself separates off a considerable portion of unwanted foreign proteins.
The precipitate is then subjected to chromatography on a basic anion exchanger, preferably on a weakly basic anion exchanger, in particular on DEAE, such as DEAE-Sepharose. In general, the active fraction elutes at an approx. 0.17 M concentration of a salt, preferably a monovalent salt such as KCl. The eluted active fraction, which has been dialyzed in a customary manner, is then subjected to an affinity chromatography, preferably on heparin, since it has been found, surprisingly, that the active fraction binds particularly well to heparin, e.g. Heparin Sepharose. The active fractions are therefore usually eluted at high salt concentrations, for example at an approx. 1.0 M concentration of a monovalent salt such as KCl. The active fractions are then subjected to chromatography on a basic anion exchanger, preferably on a strongly basic anion exchanger, in particular on Mono Q, such as Mono Q HR 16/10. The proteins are preferably eluted by means of a linear gradient, with it being possible to elute the active fractions at an approx. 10% concentration of a salt, in particular a monovalent salt such as KCl, whose concentration is approx. 1.0 M. After that, the active fractions are subjected to affinity chromatography, preferably on a dye, in particular on a blue dye, very particularly on Blue Sepharose. Preference is given to eluting the proteins using a multistep, in particular a two-step, salt gradient, with the active fractions preferably eluting well at a high salt concentration, in particular at high concentrations of a monovalent salt, very particularly at an approx. 1.0 M concentration, such as 1.0 M KCl.
According to the present invention, this is then followed by an affinity chromatography on poly(A), with it being possible to elute the active fractions at an approx. 0.35 to approx. 0.55 M concentration of a salt, in particular a monovalent salt, such as KCl. In addition, in conformity with the process according to the invention, the active fractions are subjected to chromatography on another basic anion exchanger, preferably on a strongly basic anion exchanger, in particular on Mono Q, such as SMART Mono Q. In this connection, the activity is preferably eluted at an approx. 0.17 M concentration of a salt, in particular a monovalent salt such as KCl. In conformity with the process according to the invention, the last purification step is, where appropriate, a step in which the active fractions are subjected to a gel filtration, preferably a Superdex 200 gel filtration, in particular a SMART Superdex 200 gel filtration, with it generally being possible to fractionate the active fractions satisfactorily in the presence of an approx. 0.1 M concentration of a salt, in particular a monovalent salt such as KCl.
In conformity with the process according to the invention, it is possible to purify a poly(A)-specific 3xe2x80x2-exonuclease activity approx. 600-fold with a yield of approx. 13% (see Table I). In this connection, it was particularly surprising that the poly(A) affinity chromatography in accordance with step (g) resulted in an approx. 14-fold purification of the activity according to the invention.
In another embodiment, the proteins are subjected, after the dye chromatography, to a double affinity chromatography, preferably chromatography on ssDNA Agarose followed by chromatography on 5xe2x80x2AMP Sepharose. The active fractions are normally eluted at high salt concentrations, such as an approx. 2.0 M concentration of a monovalent salt such as KCl.
The process according to the invention now results in the isolation of a poly(A)-specific 3xe2x80x2-exonuclease activity which runs at approx. 50 kDa under denaturing conditions, for example in an SDS-PAGE gel, and runs at from approx. 180 to 220 kDa under native conditions, for example on Superdex 200.
The invention therefore also relates to an approx. 50 kDa protein and, where appropriate, an associated protein (tetramer) which possesses a poly(A)-specific exonuclease activity and which can be obtained in accordance with the process according to the invention.
The present invention therefore also relates to a composition which comprises a poly(A)-specific 3xe2x80x2-exonuclease activity which can be obtained in accordance with the process according to the invention. Where appropriate, the composition comprises other additives and adjuvants.
The present invention also relates to a process for deadenylating nucleic acids, in particular for deadenylating 3xe2x80x2-located poly(A) tails belonging, preferably, to mRNA in the presence of a composition according to the invention. The deadenylation reaction preferably takes place in the presence of monovalent cations, such as K+ and/or Na+, in particular at concentrations of the monovalent cation of approx. 0.1 M. Preference is furthermore given to the deadenylation reaction taking place at a pH of approx. 7.
The present invention also relates to antibodies which react specifically with the composition according to the invention and/or a component thereof, with the composition itself being immunogenic or with it being possible to make the composition immunogenic, or to increase the immunogenicity of the composition, by coupling it to suitable carriers such as bovine serum albumin.
The antibodies are either polyclonal antibodies or monoclonal antibodies. Their preparation, which is also part of the subject matter of the present invention, is effected, for example in accordance with well-known methods, by immunizing a mammal, for example a rabbit, with the composition according to the invention, where appropriate in the presence of, for example, Freund""s adjuvant and/or aluminum hydroxide gels (see, e.g., Diamond, B. A. et al. (1981) The New England Journal of Medicine, 1344). The polyclonal antibodies which are produced in the animal due to an immunological reaction can then readily be isolated from the blood in accordance with well-known methods and, for example, purified by means of column chromatography. Preference is given to purifying the antibodies by affinity chromatography, in which, for example, the composition according to the invention has been coupled to an NHS-activated HiTrap column.
Monoclonal antibodies can, for example, be prepared in accordance with the known method of Winter and Milstein (Winter, G. and Milstein, C. (1991) Nature, 349, 293).
The present invention furthermore also relates to a pharmaceutical which comprises a composition according to the invention and, where appropriate, suitable additives or adjuvants and to a process for producing a pharmaceutical for treating cancer, autoimmune diseases, in particular multiple sclerosis or rheumatoid arthritis, Alzheimer""s disease, allergies, in particular neurodermatitis, type I allergies or type IV allergies, arthrosis, atherosclerosis, osteoporosis, acute and chronic infectious diseases and/or diabetis, and/or for influencing the metabolism of the cell, in particular in association with immunosuppression, very particularly in association with transplantations, in which pharmaceutical a composition according to the invention is formulated together with pharmaceutically acceptable additives and/or adjuvants.
Examples of suitable additives and/or adjuvants are a physiological sodium chloride solution, stabilizers, proteinase inhibitors, etc.
The present invention furthermore also relates to a diagnostic agent which comprises a composition according to the invention and, where appropriate, suitable additives and/or adjuvants and to a process for preparing a diagnostic agent for diagnosing cancer, autoimmune diseases, in particular multiple sclerosis or rheumatoid arthritis, Alzheimer""s disease, allergies, in particular neurodermatitis, type I allergies or type IV allergies, arthrosis, atherosclerosis, osteoporosis, acute and chronic infectious diseases and/or diabetis, and/or for analyzing the metabolism of the cell, in particular the immune status, very particularly in association with transplantations, in which pharmaceutical suitable additives and/or adjuvants are added to a composition according to the invention.
For example, according to the present invention, the composition according to the invention can be bound to a solid phase, e.g. consisting of nitrocellulose or nylon, and in this way be brought into contact in vitro, for example, with the body fluid to be investigated, e.g. blood, in order thereby to be able to react, for example, with autoimmune antibodies. The antibody-peptide complex can then, for example, be detected using labeled antihuman IgG or antihuman IgM antibodies. The label is, for example, an enzyme, such as peroxidase, which catalyzes a color reaction. The presence of autoimmune antibodies, and the quantity of the antibodies which is present, can thereby be determined readily and rapidly by way of the color reaction.
Another diagnostic agent comprises the antibodies according to the invention themselves. Using these antibodies it is possible, for example, to readily and rapidly investigate a human tissue sample to determine whether the composition according to the invention and/or a component thereof is present. In this case, the antibodies according to the invention are labeled, for example, with an enzyme as has already been described above. This enables the specific antibody-peptide complex to be determined readily and just as rapidly by way of an enzymic color reaction.
The present invention also relates to a test for identifying functional interactors, such as inhibitors or stimulators, comprising a composition according to the invention or antibodies according to the invention and, where appropriate, suitable additives and/or adjuvants. For this, selected substances, for example from a so-called chemical library, are employed in the deadenylation reaction which has already been described in detail above and the activity of the composition according to the invention is measured in the presence and/or absence of the substances. An example of a suitable substrate is mRNA or poly(A). The test can be carried out, for example, in analogy with the in-vitro deadenylation, as described in detail in the examples.
Another general possibility of using the composition according to the invention is therefore also that of degrading nucleic acids, in particular mRNA, in a poly(A)-specific manner. The poly(A)-specific degradation of nucleic acids can be of particular use in research laboratories.
The following tables, figures and examples are intended to clarify the invention without limiting it.